Rhizosphere-associated Pseudomonas fluorescens are known plant growth promoting (PGP) and mycorrhizal helper bacteria (MHB) of many plants and ectomycorrhizal fungi. We investigated the spatial and temporal dynamics of colonization of mycorrhizal and non-mycorrhizal Aspen seedlings roots by the P. fluorescens strains SBW25, WH6, Pf0-1, and the P. protegens strain Pf-5. Seedlings were grown in laboratory vertical plates systems, inoculated with a fluorescently labeled Pseudomonas strain, and root colonization was monitored over a period of 5 weeks. We observed unexpected diversity of bacterial assemblies on seedling roots that changed over time and were strongly affected by root mycorrhization. P. fluorescens SBW25 and WH6 stains developed highly structured biofilms with internal void spaces forming channels. On mycorrhizal roots bacteria appeared encased in a mucilaginous substance in which they aligned side by side in parallel arrangements. The different phenotypic classes of bacterial assemblies observed for the four Pseudomonas strains were summarized in a single model describing transitions between phenotypic classes. Our findings also reveal that bacterial assembly phenotypes are driven by interactions with mucilaginous materials present at roots.

Diverse communities of bacteria colonize plant roots and the rhizosphere. Many of these rhizobacteria are symbionts and provide plant growth promotion (PGP) services, protecting the plant from biotic and abiotic stresses and increasing plant productivity by providing access to nutrients that would otherwise be unavailable to roots. In return, these symbiotic bacteria receive photosynthetically-derived carbon (C), in the form of sugars and organic acids, from plant root exudates. PGP activities have been characterized for a variety of forest tree species and are important in C cycling and sequestration in terrestrial ecosystems. The molecular mechanisms of these PGP activities, however, are less well-known. In a previous analysis of Pseudomonas genomes, we found that the bacterial transportome, the aggregate activity of a bacteria's transmembrane transporters, was most predictive for the ecological niche of Pseudomonads in the rhizosphere. Here, we used Populus tremuloides Michx. (trembling aspen) seedlings inoculated with one of three Pseudomonas fluorescens strains (Pf0-1, SBW25, and WH6) and one Pseudomonas protegens (Pf-5) as a laboratory model to further investigate the relationships between the predicted transportomic capacity of a bacterial strain and its observed PGP effects in laboratory cultures. Conditions of low nitrogen (N) or low phosphorus (P) availability and the corresponding replete media conditions were investigated. We measured phenotypic and biochemical parameters of P. tremuloides seedlings and correlated P. fluorescens strain-specific transportomic capacities with P. tremuloides seedling phenotype to predict the strain and nutrient environment-specific transporter functions that lead to experimentally observed, strain, and media-specific PGP activities and the capacity to protect plants against nutrient stress. These predicted transportomic functions fall in three groups: (i) transport of compounds that modulate aspen seedling root architecture, (ii) transport of compounds that help to mobilize nutrients for aspen roots, and (iii) transporters that enable bacterial acquisition of C sources from seedling root exudates. These predictions point to specific molecular mechanisms of PGP activities that can be directly tested through future, hypothesis-driven biological experiments.

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In the terrestrial ecosystem, plant–microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram-negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Several next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. The plasmids are stably maintained during root colonization in the absence of selective pressure for more than 2 weeks.

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